Ultrasound imaging

ABSTRACT

A combined preparation comprising:  
     i) an ultrasound contrast agent capable of accumulation in tissue microvasculature; and  
     ii) a pharmacologically effective amount of a vasodilator drug  
     may be used in perfusion imaging, especially of the myocardium. The contrast agent accumulates in tissue in concentrations related to the regional rate of tissue perfusion, and the vasodilator drug enhances distinction between normally perfused and underperfused tissue.

[0001] This invention relates to ultrasound imaging, more particularlyto use of ultrasound imaging in visualising tissue perfusion, i.e. bloodflow per unit of tissue mass, in particular cardiac perfusion.

[0002] It is well known that contrast agents comprising dispersions ofgas microbubbles are particularly efficient backscatterers of ultrasoundby virtue of the low density and ease of compressibility of themicrobubbles. Such microbubble dispersions, if appropriately stabilised,may permit highly effective ultrasound visualisation of, for example,the vascular system and tissue microvasculature, often at advantageouslylow doses.

[0003] Measurements of tissue perfusion are of importance in, forexample, tumour detection, tumour tissue typically having differentvascularity from healthy tissue, and studies of the myocardium, e.g. toevaluate the blood supply thereto. Whilst contrast agent detection usingcurrent ultrasound imaging techniques may provide information as towhether particular organs or regions thereof are perfused or not, itdoes not readily permit quantification of levels of perfusion. Suchinformation, which is useful in assessing whether a patient is at riskowing to low perfusion and so may benefit from preventative methodsand/or treatment, must currently be obtained using radioisotopic imagingtechniques such as scintigraphy, positron emission tomography or singlephoton emission computed tomography. These techniques all involveinjection of radioactive substances, with potential safety risks forboth the patient and medical staff, and use of expensive imagingequipment; this inevitably prohibits their widespread use.

[0004] It is known from radionucleide cardiac imaging that patients maybe subjected to physical or pharmacological stress in order to enhancethe distinction, and thus the difference in imaging intensities, betweennormally perfused myocardium and any myocardial regions supplied bystenotic arteries. Such stress induces vasodilatation and increasedblood flow in healthy myocardial tissue, whereas blood flow inunderperfused tissue supplied by a stenotic artery is substantiallyunchanged since the capacity for arteriolar vasodilatation is alreadyexhausted by inherent autoregulation seeking to increase the restrictedblood flow.

[0005] The application of stress as physical exercise orpharmacologically by administration of adrenergic agonists may causediscomfort such as chest pains in patient groups potentially sufferingfrom heart disease, and it is therefore preferable to enhance theperfusion of healthy tissue by administration of a vasodilating drug.

[0006] The present invention is based on the finding that ultrasoundcontrast agents capable of accumulation in tissue microvasculature maybe used in perfusion imaging, especially of the myocardium, whencoadministered with a pharmacologically effective amount of avasodilating drug. Because such contrast agents will accumulate intissue in concentrations related to the regional rate of tissueperfusion, ultrasound imaging modalities such as conventional orharmonic B-mode imaging where the display is derived from return signalintensities will provide images which may be interpreted as perfusionmaps in which the displayed signal intensity is a function of localperfusion. This is in contrast to images obtained using free-flowingcontrast agents, where the regional concentration of contrast agent andcorresponding return signal intensity depend on the actual blood contentrather than the rate of perfusion of local tissue.

[0007] A disadvantage of existing radionucleide cardiac imagingtechniques is that the uptake of radionucleide tracers such as thallium201 and technetium sestamibi is limited by low contact time betweentracer and tissue and so may require maintenance of vasodilatation forthe whole period of blood pool distribution for the tracer (e.g. 4-6minutes for thallium scintigraphy) to ensure optimum effect.Accumulative ultrasound contrast agents used in accordance with thepresent invention, on the other hand, do not suffer such diffusion ortransport limitations and, especially where accumulation occurs througha process of physical entrapment, may undergo highly efficient retentionin tissue microvasculature. The period of vasodilatation needed toachieve cardiac or other perfusion imaging in accordance with theinvention may therefore be short, for example less than one minute; thiswill reduce the duration of any possible discomfort caused to patientsby administration of vasodilator drugs.

[0008] In accordance with one embodiment of the invention there isprovided a combined preparation for use as a contrast agent inultrasound perfusion imaging, especially cardiac perfusion imaging, saidpreparation comprising:

[0009] i) an ultrasound contrast agent capable of accumulation in tissuemicrovasculature, e.g. of the myocardium; and

[0010] ii) a pharmacologically effective amount of a vasodilator drug.

[0011] According to a further embodiment of the invention there isprovided a method of generating enhanced perfusion images, especiallycardiac perfusion images, of a human or non-human animal subject whichcomprises the steps of:

[0012] i) injecting an ultrasound contrast agent capable of accumulationin tissue microvasculature, e.g. of the myocardium, into the vascularsystem of said subject;

[0013] ii) coadministering a pharmacologically effect amount of avasodilator drug; and

[0014] iii) generating an ultrasound image representing perfusion of atarget organ or tissue, especially the myocardium.

[0015] Representative vasodilator drugs useful in accordance with theinvention include endogenous/metabolic vasodilators such as lactic acid,adenosine triphosphate, adenosine diphosphate, adenosine monophosphate,adenosine, nitric oxide and agents causing hypercapnia,hypoxia/hypoxemia or hyperemia; phosphodiesterase inhibitors such asdipyridamole and sildenafil; sympathetic activity inhibitors such asclonidine and methyldopa; smooth muscle relaxants such as papaverine,hydralazine, dihydralazine and nitroprusside; beta receptor agonistssuch as dopamine, dobutamine, arbutamine, albuterol, salmeterol andisoproterenol; alpha receptor antagonists such as doxazosin, terazosinand prazosin; organic nitrates, such as glyceryl trinitrate, isosorbidedinitrate and isosorbide mononitrate; angiotensin converting enzyme(ACE) inhibitors such as benazepril, captopril, enalapril, fosinopril,lisinopril, quinapril and ramipril; angiotensin II antagonists (or AT1receptor antagonists) such as valsartane, losartan and candesartan;calcium channel blockers such as amlodipine, nicardipine, nimodipine,felodipine, isradipine, diltiazem, verapamil and nifedipine;prostaglandins such as alprostadil; and endothelium-dependentvasodilators.

[0016] In view of the fact that the required vasodilatation may needonly to be short lasting, adenosine is a particularly usefulvasodilating drug, being both an endogenous substance and having a veryshort-lasting action as evidenced by a blood pool half-life of only afew seconds. Vasodilatation will accordingly be most intense in theheart, since the drug will tend to reach more distal tissues in lessthan pharmacologically active concentrations, and may result in coronaryblood flow in healthy myocardial tissue increasing by more than 400%. Itwill be appreciated that because of this short half-life, repeatedinjection or infusion of adenosine may be necessary during cardiacimaging in accordance with the invention; by way of example, an initialadministration of 150 μg/kg of adenosine may be made substantiallysimultaneously with administration of the contrast agent, followed 10seconds later by slow injection of a further 150 μg/kg of adenosine,e.g. over a period of 20 seconds.

[0017] One category of accumulative contrast agents useful in accordancewith the invention comprise gas-containing contrast agent preparationswhich promote controllable and temporary growth of the gas phase in vivofollowing administration owing to the presence of a diffusible componentcapable of inward diffusion into the dispersed gas phase to promotetemporary growth thereof, thereby acting as deposited perfusion tracers.Such compositions therefore comprise:

[0018] i) an injectable aqueous medium having gas dispersed therein; and

[0019] ii) a composition comprising a diffusible component capable ofdiffusion in vivo into said dispersed gas so as at least transiently toincrease the size thereof. Accumulative contrast agents of this type areextensively described in WO-A-9817324, the contents of which areincorporated herein by reference.

[0020] The dispersed gas in such a preparation may, for example,comprise air, nitrogen, oxygen, carbon dioxide, hydrogen, an inert gas,a sulphur fluoride, selenium hexafluoride, an optionally halogenatedsilane, an optionally halogenated low molecular weight hydrocarbon (e.g.having a molecular weight such that it is substantially or completely ingaseous form at the normal human body temperature of 37EC), a ketone, anester or a mixture of any of the foregoing. The use of perfluorinatedgases, for example sulphur hexafluoride, perfluorinated ketones,perfluorinated ethers and perfluorocarbons, including perfluoroalkanessuch as a perfluoropropane, perfluorobutane or perfluoropentane,perfluoroalkenes and perfluorocycloalkanes, may be particularlyadvantageous in view of the recognised high stability in the bloodstreamof microbubbles containing such gases.

[0021] The dispersed gas may, for example, be in the form ofmicrobubbles stabilised (e.g. at least partially encapsulated) by acoalescence-resistant surface membrane such as gelatin, a filmogenicprotein (e.g. an albumin such as human serum albumin), a polymermaterial, a non-polymeric and non-polymerisable wall-forming material ora surfactant (e.g. a phospholipid, preferably such that at least 75% ofthe surfactant material comprises molecules individually bearing netoverall charge, for example negative charge as in phosphatidylserines,phosphatidylglycerols, phosphatidylinositols, phoaphatidic acids andcardiolipins).

[0022] The diffusible component may advantageously be dispersed in anaqueous carrier liquid in the form of an oil-in-water emulsion ormicroemulsion and may, for example, comprise an aliphatic ether such asdiethyl ether; a polycyclic oil or alcohol such as menthol, camphor oreucalyptol; a heterocyclic compound such as furan or dioxane; analiphatic hydrocarbon or cycloaliphatic hydrocarbon, e.g. containing upto 7 carbon atoms; or halogenated low molecular weight hydrocarbon, e.g.contaning up to 7 carbon atoms. The use of perfluorocarbons, e.g. aperfluoroalkane such as perfluoropentane or perfluorohexane, aperfluoroalkene, a perfluorocycloalkane such asperfluorodimethylcyclo-butane, or a perfluorinated alcohol may beadvantageous.

[0023] Where the diffusible component is formulated as an emulsion itmay advantageously be stabilised using a phospholipid surfactant, e.g.as described above in connection with the stabilisation of gasdispersions.

[0024] The diffusible component may be administered by any appropriateroute, for example cutaneously, subcutaneously, intramuscularly,intravenously or by inhalation.

[0025] A further class of accumulative ultrasound contrast agent whichmay be used in accordance with the invention comprises phase shiftcolloids such as are described in WO-A-9416739, the contents of whichare incorporated herein by reference. Such agents comprise colloidaldispersions of the liquid-in-liquid type in which the dispersed liquidhas a boiling point below the body temperature of the subject to beimaged, so that it may volatilise to form expanding gas microbubblesfollowing administration. Representative examples of such agents includeemulsions of volatile hydrocarbons, particularly perfluorocarbons suchas perfluoropentane, for example stabilised with surfactants such asphospholipids, e.g. as described above in relation to emulsions ofdiffusible components.

[0026] A still further class of accumulative ultrasound contrast agentswhich may be used in accordance with the invention comprises targetableultrasound contrast agents having affinity for sites and/or structureswithin tissue microvasculature. Such targetable agents will typicallycomprise (i) a reporter moiety capable of interacting with ultrasoundirradiation to generate a detectable signal; (ii) one or more vectorshaving affinity for particular target sites and/or structures; and (iii)one or more linkers connecting said reporter and vector(s), in the eventthat these are not directly joined.

[0027] Reporters which may be useful in such targetable agents includeany of the gas-containing systems hereinbefore described in the contextof gas-containing ultrasound contrast agent formulations.

[0028] The targetable agents may, for example, comprise vectors whichhave affinity for normal or activated endothelial cells such that theytarget the vascular endothelium and become at least transientlyconcentrated on the walls of blood vessels. Activation of endotheliummay for example be caused by microbial infections, infarcts or ischemia.Representative vectors in this context include ligands for cell adhesionmolecules, for instance proteins or carbohydrate-containing molecules,as well as cell adhesion molecules themselves where these havecorresponding ligands on endothelial cell surfaces. Examples of celladhesion molecules occuring on activated endothelial cell surfacesinclude integrins, such as ICAM-1, most of which bind the Arg-Gly-Asp(RGD) amino acid sequence. Specific cell adhesion molecules may occur,or occur in elevated amounts, in relation to the formation of thrombi,for instance blood coagulation factors, e.g. such as Factor XIII, andglycoproteins such as GP IIb/IIIa on activated blood platelets. Thrombimay be targeted by platelet binding peptides such as PLYKKIIKKLLES,NDGDFEEIPEEYLQ and GPRG. Atherosclerotic plaques may be targeted byspecific peptides such as YRALVDTLK, YAKFRETLEDTRDRMY andRALVDTEFKVKQEAGAK. Damaged vessel walls may expose targetablemyocyte-specific molecules. Angiogenesis may cause elevation ofreceptors to VEGF and tumours may be targeted by cholecystokinin,alpha-melanocyte-stimulating hormone, heat stable enterotoxin 1,vasoactive intestinal peptide, α_(v)β₃ (vitronectin receptor), uPAR(urokinase plasminogen activator receptor), oncofetal fibronectin,synthetic alpha-M2 peptide from the third heavy chain complementaritydetermining region and analogues thereof. Further references to thistechnology, e.g. in targeting to fibrin, thrombi and atheroscleroticareas are found in publications by Alkanonyuksel, H et al. in J. Pharm.Sci. (1966) 85 (5), 486-490; J. Am. Coll. Cardiol. (1996) 27 (2) Supl A,298A; and Circulation, 68 Sci. Sessions; Anaheim, 13-16 Nov. 1995.

[0029] Other vectors which may be used include proteins and peptideswhich bind to cell-surface proteoglycans. Such proteoglycans, which arecomplexes of proteins and sulphated polysaccharides, are found on mostcells, including endothelial cells, and contribute to the negativesurface charge exhibited by all eukaryotic cells. This charge may beexploited in accordance with this embodiment of the invention by usingvectors which will interact electrostatically with the endothelialsurface, for example vectors comprising cationic lipids.

[0030] Linking of a reporter unit to a desired vector or vectors may beachieved by covalent or non-covalent means, usually involvinginteraction with one or more functional groups located on the reporterand/or the vector(s). Examples of chemically reactive functional groupswhich may be employed for this purpose include amino, hydroxyl,sulfhydryl, carboxyl and carbonyl groups, as well as carbohydate groups,vicinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl,imidazolyl and phenolic groups. Covalent coupling of reporter andvector(s) may therefore be effected using linking agents containingreactive moieties capable of reaction with such functional groups, e.g.as is well known in the art.

[0031] Various vectors and linking agents which it may be useful toadopt in targetable ultrasound contrast agents in accordance with thisembodiment of the invention are disclosed in EP-A-0727225 andWO-A-9640285. Suitable vectors, linkers etc. may also be selected fromthe wide range of known vectors and linking groups summarised inWO-A-9818495, WO-A-9818498, WO-A-9818500 and WO-A-9818501. The contentsof all these documents are incorporated herein by reference.

[0032] Representative ultrasound imaging techniques which may be usefulin accordance with the invention include fundamental B-mode imaging;harmonic B-mode imaging including reception of sub-harmonics and thesecond and higher harmonics; tissue Doppler imaging, optionallyincluding selective reception of fundamental, harmonic or sub-harmonicecho frequencies; colour Doppler imaging, optionally including selectivereception of fundamental, harmonic or sub-harmonic echo frequencies;power Doppler imaging, optionally including selective reception offundamental, harmonic or sub-harmonic echo frequencies; power or colourDoppler imaging utilising loss of correlation or apparent Doppler shiftscaused by changes in the acoustical properties of contrast agentmicrobubbles such as may be caused by spontaneous or ultrasound-induceddestruction, fragmentation, growth or coalescense; pulse inversionimaging, optionally including selective reception of fundamental,harmonic or sub-harmonic echo frequencies, and also including techniqueswherein the number of pulses emitted in each direction exceeds two;pulse inversion imaging utilising loss of correlation caused by changesin the acoustical properties of contrast agent microbubbles such as maybe caused by spontaneous or ultrasound-induced destruction,fragmentation, growth or coalescense; pulse pre-distortion imaging, e.g.as described in 1997 IEEE Ultrasonics Symposium, pp. 1567-1570; andultrasound imaging techniques based on comparison of echoes obtainedwith different emission output amplitudes or waveform shapes in order todetect non-linear effects caused by the presence of gas bubbles.

[0033] The following non-limitative examples serve to illustrate theinvention.

PREPARATION 1 a) Perfluorobutane Gas Dispersion

[0034] Hydrogenated phosphatidylserine (100 mg) in a 2% solution ofpropylene glycol in purified water (20 ml) was heated to 80EC for 5minutes and the resulting dispersion was allowed to cool to roomtemperature overnight. 1 ml portions were transferred to 2 ml vials, theheadspace above each portion was flushed with perfluorobutane gas, andthe vials were shaken for 45 seconds using an Espe CapMix7 mixer fordental materials, yielding milky white microbubble dispersions with avolume median diameter of 5.0 μm, measured using a Coulter Counter (allCoulter Counter measurements were made at room temperature using aninstrument fitted with a 50 μm aperture and having a measuring range1-30 μm; Isoton II was used as electrolyte).

b) Dispersion of Lyophilised Perfluorobutane Gas Dispersion

[0035] A sample of the milky white dispersion prepared as in (a) abovewas washed three times by centrifugation and removal of the infranatant,whereafter an equal volume of 10% sucrose solution was added. Theresulting dispersion was lyophilised and then redispersed in distilledwater just prior to use.

PREPARATION 2 Perfluoromethylcyclobutane Emulsion

[0036] Hydrogenated phosphatidylserine (100 mg) in purified water (20ml) was heated to 80EC for 5 minutes and the resulting dispersion wascooled to 0EC overnight. 1 ml of the dispersion was transferred to a 2ml vial, to which was added 100 μl of perfluorodimethylcyclobutane (>97%1,1-isomer, balance being 1,2- and 1,3-isomers). The vial was thenshaken for 75 seconds using a CapMix7 to yield an emulsion of diffusiblecomponent which was stored at 0EC when not in use.

EXAMPLE 1 In vivo Imaging of Dog Heart with Perfluorobutane GasDispersion and Perfluorodimethyl-cyclobutane Emulsion and CoadministeredAdenosine

[0037] An occluding snare was placed around a major branch of the leftanterior descending coronary artery of an open-chest 22 kg dog and anultrasound transit time flowmeter was placed immediately downstream ofthe occluder, which was then adjusted to produce a steady 25% flowreduction from about 14 to 10 ml/min. The contents of three syringes,respectively containing (i) an amount of a perfluorobutane microbubbledispersion prepared as in Preparation 1 corresponding to 4.4 μl of gascontent, (ii) an amount of the perfluorodimethylcyclobutane emulsionfrom Preparation 2 corresponding to 33 μl of the dispersedperfluorodimethylcyclobutane phase, and (iii) 3.0 mg adenosine dissolvedin 0.9% saline, were then intravenously injected as a simultaneousbolus; commencing 10 seconds later a further 3.0 mg of adenosinedissolved in 0.9% saline was injected slowly over 20 seconds. Imaging ofthe left ventricle of the heart was performed using an ATL HDI-3000scanner with a P5-3 probe; continuous ultrasonication at maximum powerwas applied for 1 minute to induce microbubble growth, whereafter themyocardium was examined using B-mode imaging. A clearly evidentdifference in gray scale levels could be seen between stenotic areas(brighter than baseline recordings) and normal areas (very much brighterthan baseline recordings).

EXAMPLE 2-[COMPARATIVE]

[0038] The procedure of Example 1 was repeated, but without injection ofadenosine. The differences in contrast intensity between areas suppliedby normal and stenotic arteries were now only barely visible; the maindifference from Example 1 was that the brightness of normal areas wasreduced to a level closer to that of the regions supplied by thestenotic artery.

EXAMPLE 3 Imaging with Contrast Acent Under Dobutamine Stress

[0039] Example 1 was repeated except that a continuous infusion ofdobutamine at a rate of 15 μg/kg/min was administered in place ofadenosine. After a stable dobutamine effect consisting of an increase inheart rate from the normal 100 beats per minute to 150 beats per minutewas obtained, the microbubble dispersion and the emulsion wereintravenously injected from two syringes as a simultaneous bolus.Infusion of dobutamine was continued for another 2 minutes after thecontrast agent injection. Towards the end of this period, a distinctpattern of myocardial contrast enhancement was seen, clearly depictingthe areas supplied from the stenotic artery as darker than the normalmyocardium. In addition, a myocardial contractility deficit consistingof a pronounced wall thinning was observed in the areas supplied by thestenotic artery.

What is claimed is: 1-10. (Canceled)
 11. A combined preparation for useas a contrast agent in ultrasound perfusion imaging, said preparationcomprising: i) an ultrasound contrast agent which following injectioninto the vascular system of a human or non-human animal subject willaccumulate in tissue microvasculature of said subject at concentrationsdependent on regional rates of perfusion within said tissue; and ii) avasodilatation-inducing amount of a vasodilator drug.
 12. A preparationas claimed in claim 11 wherein said vasodilator is a substance which isendogenous to said subject.
 13. A preparation as claimed in claim 12wherein said vasodilator drug is adenosine.
 14. A preparation as claimedin claim 11 wherein said ultrasound contrast agent comprises a phaseshift colloid.
 15. A preparation as claimed claim 11 wherein saidultrasound contrast agent comprises a targetable ultrasound contrastagent having affinity for sites and/or structures within tissuemicrovasculature.
 16. A preparation as claimed in claim 15 wherein saidultrasound contrast agent comprises at least one vector having affinityfor normal or activated endothelial cells.
 17. A preparation as claimedin claim 15 wherein said ultrasound contrast agent comprises at leastone protein or peptide which binds to cell-surface proteoglycans.
 18. Amethod of generating enhanced perfusion images of a human or non-humananimal subject which comprises the steps of: i) injecting into thevascular system of said subject an ultrasound contrast agent whichfollowing injection will accumulate in tissue microvasculature of saidsubject at concentrations dependent on regional rates of perfusionwithin said tissue; ii) coadministering a vasodilatation-inducing amountof a vasodilator drug; and iii) generating an ultrasound imagerepresenting perfusion of a target organ or tissue.
 19. A method asclaimed in claim 18 wherein images representative of myocardialperfusion are generated.
 20. A preparation as claimed in claim 12wherein said ultrasound contrast agent comprises a phase shift colloid.21. A preparation as claimed in claim 13 wherein said ultrasoundcontrast agent comprises a phase shift colloid.
 22. A preparation asclaimed in claim 12 wherein said ultrasound contrast agent comprises atargetable ultrasound contrast agent having affinity for sites and/orstructures within tissue microvasculature.
 23. A preparation as claimedin claim 13 wherein said ultrasound contrast agent comprises atargetable ultrasound contrast agent having affinity for sites and/orstructures within tissue microvasculature.
 24. A preparation as claimedin claim 11 wherein said ultrasound contrast agent comprises: i) aninjectable aqueous medium having gas dispersed therein, said dispersedgas having affinity for sites and/or structures within tissuemicrovasculature; and ii) a composition comprising a diffusiblecomponent, said diffisible component being separate from said dispersedgas and being capable, following administration of said contrast agentto said subject, of diffusion in vivo into said dispersed gas so as topromote controllable growth and temporary retention of said dispersedgas within tissue microvasculature in said subject.